Thermodynamic studies of complexes in Cu(II)/Uridine- 5’-diphosphoglucuronic acid system

Thermodynamic studies of complexes in Cu(II)/Uridine- 5’-diphosphoglucuronic acid system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Thermodynamic studies of complexes in Cu(II)/Uridine- 5’-diphosphoglucuronic acid system Klaudia Stachowiak, Michal Zabiszak, Jakub Grajewski, Anna Teubert, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4184106/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Binary system of uridine-5'-diphosphoglucuronic acid with copper(II) ions have been studied. Potentiometric studies in aqueous solutions using computer data analysis were carried out. The pH of dominance, the overall stability constants (log β ) and the equilibrium constants of the formation reaction (log K e ) were determined for each complex compound formed in the studied system. Spectroscopic studies were carried out to determine the mode of coordination in the compounds studied. Biological analysis of the compounds obtained showed an increase in the biological activity of the complexes tested against the free ligand. Biological sciences/Biochemistry/Bioinorganic chemistry Biological sciences/Biochemistry/Biophysical chemistry Biological sciences/Biochemistry/Chemical modification Biological sciences/Biochemistry/Dna Biological sciences/Biochemistry/Metals Biological sciences/Biochemistry Biological sciences/Chemical biology Physical sciences/Chemistry copper(II) ions uridine derivatives potentiometric measurements spectroscopic studies biological studies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Glycans are compounds that play an important role in living organisms. Changes in their concentration and differences in their structure on the surface of cells are important for diagnosing developing cancer or infection in the body. Compounds that regulate the level of glycans in the organism are sugar derivatives of uridine diphosphate. These derivatives also play a key role in the synthesis of glycans. Due to their properties, these compounds may constitute a new group of anticancer drugs [ 1 – 6 ]. Metal ions belong to a group of elements that are essential for the proper functioning of organisms. They play an important role in the stabilization of protein structures and are an active center of many enzymes [ 7 , 8 ]. An important trace element in living organisms is the copper(II) ion, which can be found in the brain, liver, or bones. Copper(II) ions are part of many enzymes, including cytochrome c oxidase, peroxidase, tyrosinase, and lysil oxidase. Furthermore, these ions are involved in the synthesis of hemoglobin and melanin [ 9 – 11 ]. Copper(II) ion concentration disorders can cause diseases such as anemia, osteoporosis, Parkinson’s disease, hernias, Menkes syndrome, Hodgkin’s disease, leukemia, and general weakness [ 9 , 10 , 12 ]. Nucleic acids are biomolecules that play an important role in living organisms. These compounds are involved in the transcription, transmission, and storage of genetic information and also constitute the genetic material of a cell. Nucleotides are the basic unit that builds nucleic acids. A nucleotide consists of a nucleobase, five-carbon sugar (ribose or 2- deoxyribose), and a phosphate group. Depending on the origin of the amino acids, nucleotides can be divided into purine and pyrimidine nucleotides [ 13 – 15 ]. Nucleosides, such as nucleotides, in their structure contain amino acids and sugar, ribose or deoxyribose. Both play a key role in the living organism: they participate in the synthesis of lipids and the metabolism of carbohydrates and are components of many coenzymes [ 16 , 17 ]. One of the representatives of pyrimidine nucleosides is uridine, which is composed of sugar ribose and uracil. It is necessary in the process of RNA and glycogen synthesis and is a precursor of uridine phosphates. Uridine and its derivatives play an important role in the functioning of the central nervous system. Uridine-5'-triphosphate (UTP) is used during glycogen synthesis to produce uridine-5’-diphosphate glucose (UDP-glucose) [ 18 – 22 ]. Nucleic acid bases can exist in several different forms of tautomerism. In molecules of nucleic acid bases, keto-enol and amino-imino tautomerism can occur. The formation of tautomeric forms is related to the presence of protons capable of migration in the molecules of these organic substances. The appearance of tautomeric forms is important, e.g. in pharmacy, chemistry, and physics. In addition, the formation of different tautomeric forms can influence the structural and chemical diversity of these compounds and thus their biological function. Therefore, replacing one nuclear base with its tautomer can result in an incorrect base pair, causing an error in the genetic code or a mutation [ 23 – 25 ]. D-glucuronic acid (GluA) is a derivative of glucose and is classified as uronic acids. In its molecular components, it includes the carboxyl, hydroxyl, and pseudo-aldehyde group. D-glucuronic acid is obtained in the process of dehydrogenation of UDP-glucose [ 26 – 28 ]. These compounds play an important role in the glucuronidation process. This process allows the removal of compounds such as bilirubin, xenobiotics, and steroid hormones from the organism [ 26 , 29 ]. The presence of two functional groups, carboxyl and hydroxyl, in the D-glucuronic acid molecule promotes the formation of metal-ligand complex compounds. Ligands such as D-glucuronic acid can be used as masking agents for various metals, including toxic metals [ 28 , 30 , 31 ]. Uridine-5’-diphosphoglucuronic acid (UDP-GluA) is a derivative of uridine-5’-diphosphate and D-glucuronic acid and occurs in the liver and kidneys [ 32 ]. It’s synthesis is catalyzed by UDP-glucose dehydrogenase. Uridine-5’-diphosphoglucuronic acid plays an important role in reactions that are catalysed by UDP-glucuronosyltransferase. UDP-glucuronylotransferases are a group of enzymes involved in the detoxification process of the living organism. These enzymes catalyze the glucuronidation of potentially toxic and carcinogenic metabolic products such as cannabinoids and such compounds as thyroxin, some bile acids, morphine and acetaminophen. [ 33 ]. The following article presents the results of potentiometric and spectroscopic studies on the formation of complex compounds in the system of uridine-5 '-diphosphoglucuronic acid and copper(II) ions. The internal coordination sphere in the obtained complexes was also determined. Furthermore, the antitumor properties of the obtained complexes were investigated. Results and discussion Binary system of Copper(II) Ion/Uridine-5’-diphosphoglucuronic acid The analysis of the potentiometric studies confirmed the presence of three protonated forms of the ligand: (UDP-GluA)H, (UDP-GluA)H 2 , and (UDP-GluA)H 3 in the system studied. The protonation constants of these forms and their formation reactions are given in Table 1 . The structural formula of the studied ligand with a highlighted potential coordination site is shown in Fig. 1 . In the pH range studied, the carboxylic group of D-glucuronic acid, the nitrogen atom in the uridine ring, and one of the phosphate residues are deprotonated and considered potential coordination centers. Table 1 The protonation constant (log β ) of uridine-5’-diphosphoglucuronic acid, the equilibrium constant of formation (log K e ) (standard deviations are given in parentheses). Species log β ; [ 34 ] log K e Reaction (UDP-GluA)H 8.66(2); 9.40 [ 34 ] 8.64 (UDP-GluA) 4− + H + ↔ (UDP-GluA)H 3− (UDP-GluA)H 2 11.67(4); 12.79 [ 34 ] 3.01 (UDP-GluA)H 3− + H + ↔ (UDP-GluA)H 2 2− (UDP-GluA)H 3 13.85(5); 14.19 [ 34 ] 2.17 (UDP-GluA)H 2 2− + H + ↔ (UDP-GluA)H 3 − The deprotonation of the -O-PO 3 H − group (log β = 13.82, log K e =2.17) and the -COOH group (log β = 11.65, log K e =3.01) starts at a pH value lower than the study range. The partially protonated form occurs in the system up to a pH value of about 5,5. The deprotonation process of the proton of -N(3)H (log β = 8.64, log K e =8.64) begins below the test scale. This form is present in solution up to a pH of about 9.5 and at its maximum represents approximately 100% of all forms in the system. At pH above 9.5, the system is dominated by the fully deprotonated form of the ligand (Fig. 2 ). At high pH values, when a proton from -N(3)H is deprotonated, lactam-lactim tautomerism occurs due to the alkaline medium. As a result of tautomerism, the free electron pair from the nitrogen atom migrate, causing the formation of a double bond between the C4 carbon atom and the nitrogen atom. The formation of the double bond causes an electron pair to leave the bond between the carbon atom C4 and the oxygen atom and migrates this pair to the oxygen atom (Fig. 3 ). Computer analysis of the potentiometric measurements confirmed the presence of two protonated forms and two hydroxocomplexes: Cu(UDP-GluA)H 2 , Cu(UDP-GluA)H, Cu(UDP-GluA)(OH) and Cu(UDP-GluA)(OH) 3 . The stability constants (log β ), equilibrium constants of formation (log K e ), and examples of complex formation are presented in the table (Table 2 ). Table 2 The overall stability constants (log β ) and the equilibrium constants of formation (log K e ) of the complexes formed in the studied system (standard deviations are given in parentheses). Species log β log K e Reaction Cu(UDP-GluA)H 2 15.61(3) 3.94 Cu 2+ + (UDP-GluA)H 2 ↔ Cu(UDP-GluA)H 2 Cu(UDP-GluA)H 12.52(2) 3.86 Cu 2+ + (UDP-GluA)H ↔ Cu(UDP-GluA)H Cu(UDP-GluA)(OH) -0.78(2) 12.98 Cu 2+ + (UDP-GluA) + H 2 O ↔ Cu(UDP-GluA)(OH) + H + Cu(UDP-GluA)(OH) 3 -20.87(3) 7.45 Cu(UDP-GluA)(OH) + 2H 2 O ↔ Cu(UDP-GluA)(OH) 3 + 2H + The correctness of the assumed model was determined by comparing the theoretical computer-generated curve and the experimental curve (Fig. 4 ). The experimental and theoretical curves correspond practically over the entire range, which proves the correctness of the assumed model. The sigma parameter for the adopted model is: 12.405. Complex Cu(UDP-GluA)H 2 starts forming at a pH below 2.5 (Fig. 5 ). This form dominates at pH value approximately 2.5 and binds about 55% of the cooper(II) ions. Cu(UDP-GluA)H started forming at pH value below 2.5. The protonated form dominates at pH 5.0-5.5 and binded maximally to 70% of copper(II) ions. The first hydroxy complex Cu(UDP-GluA)(OH) started to form at pH 5.5 and dominated at pH values about 8.0, where it binded approximately 65% of Cu 2+ . At pH 8.0, the las complex form started forming: Cu(UDP-GluA)(OH) 3 . This complex dominated at pH value above the test range. At pH about 10.5 this hydroxy complex binding about 55% of copper(II) ions. Spectroscopic Studies UV-vis and EPR spectroscopy Spectroscopic methods were used to analyze the forming complex compounds. UV-Vis and EPR measurements were performed at pH values that provided the highest possible percentage of a given form. These pH values were selected on the basis of distribution curves. The obtained spectroscopic parameters are summarized in Table 3 . Table 3 UV-Vis and EPR spectroscopic parameters for the formation of complex forms. Species pH g ǁ A ǁ [cm − 1 ] λ max [nm] ε [M − 1 cm 1 ] Absorbance chromophore Cu(UDP-GluA)H 2 2.5 2.39 136∙10 − 4 810 23 0.023 {1O} Cu(UDP-GluA)H 5.0 2.37 145∙10 − 4 800 30 0.030 {1O} Cu(UDP-GluA)(OH) 8.0 - - 710 93 0.093 {1N, 2O} Cu(UDP-GluA)(OH) 3 10.5 - - 690 112 0.112 {1N, 3O} The change in the internal coordination sphere in complexes containing copper(II) ions is associated with a shift in absorbance toward lower wavelengths (Fig. 6 ). For the Cu(UDP-GluA)H 2 and Cu(UDP-GluA)H complexes, coordination occurs through one oxygen atom derived from the glucuronic acid moiety or phosphate group. With increasing pH values, nitrogen in the UDP-GluA molecule is deprotonated and a coordination bond is formed with this donor atom. For the Cu(UDP-GluA)(OH) complex, coordination occurs through a nitrogen atom derived from the UDP molecule and two oxygen atoms derived from the glucuronic acid molecule and phosphate groups of the UDP molecule. The internal coordination sphere of the last complex form from the studied system includes a nitrogen atom N(3) derived from the UDP molecule and three oxygen atoms derived from carbonyl group -COOH of glucuronic acid and two phosphate residues -O-PO 2 H. On the basis of EPR spectra analysis, the formation of monomeric complex forms in the studied system was confirmed, and characteristic spectra for copper(II) ions were observed (Fig. 7 ). The spectral parameters g ǁ = 2.39 and A ǁ = 136∙10 − 4 cm − 1 for the Cu(UDP-GluA)H 2 complex and g ǁ = 2.37 and A ǁ = 145∙10 − 4 cm − 1 for the Cu(UDP-GluA)H complex indicate the participation of one oxygen atom in the formation of the coordination bond. The EPR spectra obtained are analogous due to the similar coordination mode in the compounds analysed. CD spectroscopy The first series of CD measurements was carried on uridine-5’-diphosphoglucuronic acid at different pH to exclude protonation/deprotonation effect on the conformation of the ligand. Based on the results of potentiometric studies, pH 3.0 and 10.0 were chosen. The results of these measurements show that there is no significant change in CD spectra in the range of 250–280 nm which originates from the absorption of uridine base. The maximum observed for the acidic solution is ∆ε = 3.81 at 269 nm and ∆ε = 4.29 at 266 for the basic one. In the short-wave part of the spectra a negative Cotton effect can be observed for the sample in the basic solution. This result is usually related [ 35 , 36 ] to deprotonation of chiral carboxylic acid, which shifts the n-π* transition and CD maxima towards shorter wavelengths (∆ε = -0.84 at 228 for the acidic solution nm and ∆ε = -2.14 at 216 for the basic one). The loaded CD spectra for both solutions are presented in Fig. 8 and indicate the conformation of uridine-5’-diphosphoglucuronic acid is not pH dependent in water solutions. As the uridine-5’-diphosphoglucuronic acid have many potential electron donor sites the possible coordination of Cu(II) ions may take place by different parts of the ligand causing its conformational changes. These coordination modes may also vary due to pH changes. The CD measurements of Cu(II)/UDP-GluA system were performed at a pH that was previously determined on the basis of potentiometric measurements knowing that the main form of the complex is usually present alongside other minor ones. In the case of measurements for all pH, the same sequence of Cotton effects is observed with a positive long-wave effect located in the range of 262–272 nm and negative Cotton effects located in the range of 218–239 nm. The main Cotton effects at a given wavelength are summarized in the Table 4 . Table 4 Cotton effects for Cu(II)/UDPGluA system in water solutions. pH 2.5 5.0 8.0 10.5 ∆ε (nm) 2.98 (272) 2.91 (267) 2.85 (267) 4.21 (262) -0.95 (239) -0.85 (239) -0.70 (239) -0.98 (237) -1.01 (224) -1.11 (228) -0.19 (227) -1.94 (227) -1.15 (219) -0.50 (219) -2.70 (218) The results indicates no substantial changes in the ligand conformation in the pH range of 2.5-8.0. The spectrum measured at pH = 10.5 has more intense and blue shifted maximum which is in agreement with NMR shifts measurements and may be associated with the change of donor atoms in the system. Additionally this change can be also connected with the shift of equilibrium of base-promoted lactam-lactim tautomerization, which is also observed in the shift of C4 signal in the 13 C NMR spectra. The negative Cotton effects in the range of 218–239 nm are derived from the interaction of the carboxylic acid residue with the chiral environment. As expected, there is a general tendency to increase the intensity of these effects and shift towards shorter wavelengths with increasing pH, however, the mutual proximity of positive Cotton effects of relatively high intensity may affect their height and location on the CD spectrum, which makes their precise interpretation difficult. CD spectra of the Cu(II)/UDP-GluA system are presented in Fig. 9 . NMR spectroscopy NMR studies were conducted to determine the mode of coordination in the studied system. The studies were performed for ligand and complexes for two pH values. The results obtained are summarized in Table 5 . Table 5. Differences between 13 C NMR and 31 P NMR chemical shifts form the ligand in the Cu(UDP-GluA) system in relation to the free ligand [ppm]. System pH (UDP-GluA) C2 C4 C5 C6 C1’ C2’ C3’ C4’ C5’ C6’ C7’ C8’ C9’ C10’ C11’ P1 P2 Cu(UDP-GluA)H 5.0 0.06 -0.03 0.06 0.01 0.06 0.0 0.47 0.01 0.11 0.20 0.24 0.10 0.00 - 0.01 - 7.93 Cu(UDP-GluA)(OH) 8.0 -0.05 -0.91 -0.02 0.07 0.36 0.03 0.13 0.02 0.49 -0.70 0.38 0.12 0.29 - 0.09 0.16 4.5 Analysis of 13 C NMR and 31 P NMR spectra revealed changes in chemical shifts between the free ligand and the complex compound. At low pH values, changes in chemical shifts were observed in carbon C6 ’ (0.20) derived from the glucuronic acid moiety and at the phosphorus atom P2 (7.93). At these pH values, coordination occurs between the oxygen atom of glucuronic acid or the oxygen atom of the phosphate group of the UDP moiety. This type of coordination occurs for the complex Cu(UDP-GluA)H. At higher pH values, changes in chemical shifts were observed at the C4 (-0.91) carbon atom and P2 (4.5) phosphorus atom of the UDP molecule and C6 ’ (-0.70) carbon atom of glucuronic acid moiety. These changes are due to deprotonation of nitrogen atom at higher pH values and the occurrence of lactam-lactim tautomerism. These changes explain the chemical shift of only the C4 carbon and not the C4 and C2 carbons in the 13 C NMR spectrum. At pH 8.0, coordination occurs through an oxygen atom located at the C4 carbon in the uridine ring and through one of the oxygen atoms of the phosphate group and an oxygen atom derived from the carbonyl group of D-glucuronic acid. A schematic of the lactam-lactim tautomerism is shown in Fig. 10 . Cytotoxicity The IC50 (half maximal inhibitory concentration) is defined as the concentration of compound needed to inhibit a biological process or response by 50%. The metabolic activity of the cells after 24 h incubation with tested compounds at pH5 was similar (Fig. 11 ). The same for IC50 values (Table 6 ). However after 72h higher cytotoxicity could be observed for Cu UDP-GluA. A549 cells reduced their activity by 44,4% in comparison to control, e.g. cells cultured in standard growth medium. In turn, after 72h incubation with UDP-GluA at pH8 no changes in cells’ metabolic activity could be detected, therefore IC50 value could not be calculated. Cu(UDP-GluA) at pH8 after 72h incubation reduced the A459 cell’s metabolic activity by 52,2%. However, the effect was strongly time-dependent. Table 6 IC50 values [uM] calculated based on the MTT assay results. Species 24h 72h (UDP-GluA) pH5 0.317 ± 0.006 0.781 ± 0.005 Cu(UDP-GluA) pH5 0.377 ± 0.005 0.116 ± 0.005 (UDP-GluA) pH8 0.295 ± 0.004 n.d.* Cu(UDP-GluA) pH8 1.719 ± 0.01 0.094 ± 0.004 n.d. – not detectable, to low cytotoxicity to calculate LC50. Conclusions The formation of complexes in the binary system of uridine-5'-diphosphoglucuronic acid and copper(II) ions has been established. The existence of two types of complex forms in the system was observed: MHxL and ML(OH)x. Depending on the pH, different coordination modes were observed in the obtained complex compounds. The types of chromophores were determined by UV-Vis, EPR and NMR spectroscopic studies. It was observed that, at low pH values, coordination occurs through an oxygen atom derived from the glucuronic acid molecule or phosphate group. As the pH increases, the nitrogen atom in the ligand molecule is deprotonated and a coordination bond is formed with this donor atom. Oxygen atoms derived from UDP phosphate residues also play an important role in the coordination process. The bioassays carried out showed an increase in the biological activity of the complex compounds tested against the free ligand. As the incubation time increases, the biological activity of the tested compounds increases. Methods Materials Uridine-5’-diphosphoglucuronic acid trisodium salt was obtained from Sigma-Aldrich and copper (II) nitrate was obtained from Merck. All of these materials were used without purification. Potentiometric study Potentiometric titration was performed using a Titrando 713 Methrom equipped with an autoburette with a Methrom 6.0233.100 combined glass electrode calibrated prior to each test. Calibration was performed prior to each titration with two buffer solutions of pH 4.002 and pH 9.225. All measurements were carried out under strictly defined conditions of constant ionic strength of 0.1 M KNO 3 , temperature 20 ± 1°C, an inert gas atmosphere helium (He 5.0 Ultra High Purity; 9.1 m 3 ; 200 bar) and a pH range of 2.5 to 11.0. The metal to ligand ratio was 1:1 and the concentration of ligands and copper(II) ions [Cu 2+ ] was 0.001 mol/dm 3 . The protonation constant and the stability constant of the complex were determined using the HYPERQUAD 2008 programme. The calculations allowed us to determine the model of complex formation in the systems studied. The correctness of the assumed model was verified by analysing the standard deviations and the convergence of the experimental and theoretical curves [ 37 ]. At a pH of around 8, each sample began to take on a light blue colour, and a small amount of precipitate was observed when the samples were discarded. UV-vis spectroscopy UV-Vis spectroscopy studies were performed on the SHIMADZU UV-1900 spectrophotometer using the UVProbe programme. Measurements were made in the wavelength range of 550 to 900 nm. The concentration of the metal ion was 0.001 mol/dm 3 and the metal to ligand molar ratios were 1:1. The data obtained with UVProbe were saved as a text file, and the UV-Vis spectra were then produced with SigmaPlot 11.0. EPR spectroscopy The EPR spectra were carried out at a temperature of -196°C, using glass capillary tubes, and recorded on an SE/X2457 Radiopan spectrometer. EPR studies were performed for copper (II) ion systems, in which the concentration of metal ions was 0.005 M in a solution of water:glycol 3:1 and the metal ligand ratio was 1:1. NMR spectroscopy 13 C and 31 P NMR spectroscopic investigations were performed for the ligand and complex forms formed at two pH values: pH = 5.0 and pH = 8.0. Samples were prepared by dissolving the corresponding reactants in a deuterated solvent and the pD values of each prepared sample were determined by NaOD and DCl taking into account the relation pD = pH + 0.4 [ 38 ]. The concentration of ligands in the samples was 0.05 mol/dm 3 and the M:L ratio was 1:100. CD spectroscopy The CD and corresponding UV spectra were recorded on a JASCO J810 spectropolarimeter at ambient temperature. Spectra were recorded in the range of 185–400 nm in water solutions and accumulated with four scans for both UDPGluA and its copper(II) complexes. Water for the experiments was extra purified by Merck Millipore Simplicity UV apparatus to lower the absorbance especially in the short-wave part of the measuring range. The measurements were performed in N 2 gas atmosphere (flow 10 L/min) and optical pathlength was 0.1 cm. Concentrations of measured solutions were 1 x 10 − 4 M, which allowed the absorbance to be maintained at an acceptable level. Cell line and cell culture The A549 cell line was initially initiated through an explant culture of lung carcinoma tissue from a 58-year-old male. The cell line was purchased from ATCC: the Global Bioresource Center. Cells are hypotriploid with the modal chromosome number of 66 in 24% of cells. As verified at ATCC, there are six markers present in single copies in all cells, e.g. der(6)t(1;6) (q11;q27); del(6) (p23); del(11) (q21), del(2) (q11), M4 and M5. Most cells have doubled sex chromosomes, single copies of N2 and N6 chromosomes, and four copies of N12 and N17 chromosomes. Basic cell culture reagents and other materials were purchased from Corning. A549 cells were routinely cultured in F-12K medium supplemented with a fetal bovine serum to a final concentration of 10%. They were maintained at 37°C in a humidified cell culture incubator (Thermo Fisher Scientific) with 5% CO 2 at a concentration between 6x10 3 and 6x10 4 cells per cm 2 . The standard growth medium was changed two or three times a week. MTT assay The assay allows for the detection of the cells’ metabolic activity. It is based on the intracellular reduction of the water-soluble MTT reagent to an insoluble formazan by respiring cells. A549 cells were detached from the growth surface, counted and transferred to 96-well plates at a density of 4.5x10 3 cells per cm 2 . After 24h preincubation in standard conditions, tested compounds were added, and cells were cultured for 24 or 72 h. The medium was discarded, and cells were incubated with the MTT solution (1mg/ml) for 1 h at 37°C. Formed purple formazan crystals were dissolved in DMSO (StanLab). The absorbance was read at OD 570 nm using a microplate reader Multiskan Sky (Thermo Fisher Scientific). Declarations Author contributions statement Conceptualization, K.S. and M.Z.; methodology, K.S., M.Z., J.G. and R.J; validation, M.Z and R.J.; formal analysis, M.Z. and J.G.; investigation, K.S., J.G., A.T. and A.B.; writing—original draft preparation, K.S.; visualization, K.S.; supervision, R.J., M.Z. and K.S. All authors reviewed the manuscript. Competing interests The authors declare no conflict of interest. Author Contribution Conceptualization, K.S. and M.Z.; methodology, K.S., M.Z., J.G. and R.J; validation, M.Z and R.J.; formal analysis, M.Z. and J.G.; investigation, K.S., J.G., A.T. and A.B.; writing—original draft preparation, K.S.; visualization, K.S.; supervision, R.J., M.Z. and K.S. All authors reviewed the manuscript. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Data Availability Data is provided within the manuscript or supplementary information files References A., Varki, Glycobiology, 2017, 27, 1, 3-49, doi: 10.1093/glycob/cww086 Fuster, M. M., & Esko, J. D. (2005). The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 5(7), 526-542. doi:10.1038/nrc1649 Dube, D. H. & Bertozzi, C. R. (2005). Glycans in cancer and inflammation - Potential for therapeutics and diagnostics. Nature Reviews Drug Discovery, 4(6), 477–488. doi:10.1038/nrd1751 Schachter, H., Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides, Biochemistry and Cell Biology, 64(3), 163–181, 1986 Dennis J., W., Nabi I., R., Demetriou M., Metabolism, Cell Surface Organization, and Disease, Cell, 139(7): 1229–1241, 2009 Brewer C., F., Miceli M., C., Baum L., G., Clusters, bundles, arrays, and lattices: novel mechanisms for lectin–saccharide- mediated cellular interactions, Current Opinion in Structural Biology, 12(5), 616–623, 2002 Moustakas, M. The role of metal ions in biology, biochemistry and medicine, Materials, 2021, 14, 549, 2-4, doi: 10.3390/ma14030549 Oves, M., Saghir Khan, M., Huda Quari, A., Nadeen Felemban, M., Almeelbi, T., Journal of Bioremediation and Biodegradation, 2016, 7, 2, 1-15, doi: 10.4172/2155-6199.1000334 Angelova, M., Asenova, S., Nedkova, V., Koleva-Kolarova, R., Tarika Journal of Science, 2011, 9, 88-98 Osredkar, J., Sustar, N., Journal of Clinical Toxicology, 2011, 1-18, doi:10.4172/2161-0494.S3-001 Hoffmann, S., K., Goslar, J., Bregier-Jarzebowska, R., Gasowska, A., Zalewska, A., Lomozik,L., Journal of Inorganic Biochemistry, 2017, 177, 89-100, doi: 10.1016/j.jinorgbio.2017.09.004 Horodyjewska, A., Popioek,., Kocot, J., Biometals, 2014, 27, 611-621, doi: 10.1007/s10534-014-9736-5 Kaczkowski, J., Podstawy Biochemii, Wydawnictwo naukowe PWN, Warszawa, 2017 Kumar, A., Kumar, D., Journal of Molecular Structure, 2020, 1222, 1-12, doi.org/10.1016/j.molstruc.2020.128889 Belmont, P., Constant, J., F., Demeunynck, M., The Royal Society of Chemistry, 2001, 30, 70-81, doi.org/10.1039/A904630E Lomozik, L., Jastrzab, R., Journal of Solution Chemistry, 2007, 36, 357-374, doi: 10.1007/s10953-006-9114-1 Saenger, W., Angewandte Chemie International Edition, 1973, 12, 591-682, doi.org/10.1002/anie.197305911 Yamamoto, T., Koyama, H., Kurajoh, M., Shoji, T., Tsutsumi, Z., Moriwaki, Y., Clinica Chimica Acta, 2011, 412, 1712-1724, doi.org/10.1016/j.cca.2011.06.006 Dobolyi, A., Juhasz, G., Kovacs, Z., Kardos, J., Current Topics in Medicinal Chemistry, 2011, 11, 1058-1067, doi: 10.2174/156802611795347618 Cansev, M., Brain Research Reviews,2006, 52, 389-397, doi.org/10.1016/j.brainresrev.2006.05.001 Berg, J., M., Tymoczko, J., L., Stryer, L., Biochemistry, W.H. Freeman and Company, 2002 Zhang, N., Yin, Y., Xu, JS, Chen, WS, Molecules, 2008, 13, 1551-1569, doi: 10.3390/molecules13081551 Wojnarowska, Z., Paluch, M., Wlodarczyk, P., Dulski, M., Wrzalik, R., Roland, C., M., The Journal of Pchysical Chemistry Letters, 2012, 3, 2288-2292, doi: 10.1021/jz300541t Fedeles, B., I., Li, D., Singh, V., Frontoers in Molecular Biosciences, 2022, 8, 1-13, doi: 10.3389/fmolb.2021.823253 Singh, V., Fedeles, B., I., Essigmann, J., M., Role of Tautomerism inRNA Biochemistry, 2014, 21(1), 1-13, doi: 10.1261/rna.048371.114 Vina, I., Linde, R., Patetko, A., Sejmonows, P., IJRRAS, 2013, 14, 2, 217-230, www.arpapress.com/Volumes/Vol14Issue2/IJRRAS_14_2_02.pdf Dutton, G., J., Glucuronic Acid Free and Combined, Academic Press New York and London, 1966 Jastrzab, R., Nowak, M., Skrobanska, M., Zabiszak, M., Journal of Coordination Chemistry, 2016, 1-19, doi: 10.1080/00958972.2016.1205738 Vina, I., Sejmonows, P., Linde, R., Patetko, A., IJRRAS, 2013, 14, 1, 17-25, www.arpapress.com/Volumes/Vol14Issue1/IJRRAS_14_1_02.pdf Richel, A., Laurent, P., Wathelet, B., Wathelet, J., Paquot, M., Tetrahedrol Letters, 2010, 51, 1356-1360, doi.org/10.1016/j.tetlet.2009.12.065 Ferrari, E., Grandi, R., Lazzari, S., Saladini, M., Journal of Inorganic Biochemistry, 2005, 99, 2381-2386, doi: 10.1371/journal.pone.0057937 Capiello, M., Giuliani, L., Rane, A., Pacifici, G., M., European Journal of Drug and Pharmacokinetics, 2000, 25, 161-164, doi: 10.1007/BF03192308 Hauser, S., C., Ziurys, J., C., Gollan, J., L., Biochemica et Biophysica Acta, 1988, 967, 149-157, doi: 10.1016/0304-4165(88)90004-9 Jastrzab, R., Lomozik, L., Coordination mode in the binary systems of copper(II)/O-phospho-L-serine. Journal of Coordination Chemistry, 2009, 62(5), 710–720. doi:10.1080/00958970802317855 Glasoe, P.K.; Long, F.A. Use of glass electrodes to measure acidities in deuterium oxide. J. Phys. Chem. 1960, 64, 188–190, doi:10.1021/j100830a521. E., Garribba, E., Lodyga-Chruscinska, D., Sanna, G., Micera, Inorganica Chimica Acta, 2001, 322(1-2), 87-98, doi:10.1016/s0020-1693(01)00554-0 Kacprzak, K., Grajewski, J., Gawronski, J., Tetrahedron: Asymetry; Indicator displacement sensor for efficient determination of α-hydroxydicarboxilic acids and their chiral discrimination; 2006, 17 (9), 1332-1336; doi: 10.1016/j.tetasy.2006.03.037 Hoffmann, M., Grajewski, J. Gawronski, J., New Journal of Chemistry, Extending the applications of circular dichroism in structure elucidation: aqueous environment breaks the symmetry of tartrate dianion, 2010, 34 (9), 2020-2026, doi: 10.1039/C0NJ00072H Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4184106","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":298005964,"identity":"8ff475ca-2364-4547-a8c8-82705efe203d","order_by":0,"name":"Klaudia Stachowiak","email":"","orcid":"","institution":"Adam Mickiewicz University in Poznan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Klaudia","middleName":"","lastName":"Stachowiak","suffix":""},{"id":298005967,"identity":"7dbcc621-c5bf-49a8-b2e7-71de009475b4","order_by":1,"name":"Michal Zabiszak","email":"","orcid":"","institution":"Adam Mickiewicz University in Poznan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michal","middleName":"","lastName":"Zabiszak","suffix":""},{"id":298005970,"identity":"bcbba8d5-0a0a-4f70-98e7-ac611c621071","order_by":2,"name":"Jakub Grajewski","email":"","orcid":"","institution":"Adam Mickiewicz University in Poznan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jakub","middleName":"","lastName":"Grajewski","suffix":""},{"id":298005973,"identity":"3e9fef95-4450-424b-8263-1300c808cd99","order_by":3,"name":"Anna Teubert","email":"","orcid":"","institution":"Polish Academy of Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Teubert","suffix":""},{"id":298005976,"identity":"541763c8-06d8-4c49-b5c7-e42f1088770b","order_by":4,"name":"Anna Bajek","email":"","orcid":"","institution":"Nicolaus Copernicus University in Torun","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Bajek","suffix":""},{"id":298005978,"identity":"7309bd7c-423c-42f4-ab7f-95a21c10ae66","order_by":5,"name":"Renata Jastrzab","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYPACZjkIzQbE7MRoOMDAbIzQwkyklsQGorWYt/ce/Pyhxjq9f9oZA4YPZYcZzAlpkTlzLlniwLH03Bm3cwwYZ5w7zGDZTECLhESOgcQBtsO5DUAtzLxthxkMDhPSIv/G+MeBf4fT5UFa/hKlRYLHTOJg2+EEA5AWRqK08OSYWZztSzfceDut4GDPuXQewn5hP2N8o+Kbtbzc7eSND36UWcuZszcQ0IMMDgAxjwEJGqCADC2jYBSMglEwzAEAKQZA5Vx1TpkAAAAASUVORK5CYII=","orcid":"","institution":"Adam Mickiewicz University in Poznan","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Renata","middleName":"","lastName":"Jastrzab","suffix":""}],"badges":[],"createdAt":"2024-03-28 18:50:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4184106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4184106/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55977577,"identity":"67540363-683b-4d79-b3c7-434e53295e3a","added_by":"auto","created_at":"2024-05-07 05:55:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25472,"visible":true,"origin":"","legend":"\u003cp\u003eFormula of the uridine-5’-diphosphoglucuronic acid\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/4cd7c57a65faa069c388c000.png"},{"id":55976716,"identity":"1061b558-f5d7-4b28-93a9-2dd02055a845","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40885,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution diagram of the protonation of UDPGluA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/fd2bff1a57c692ddfdb4659a.png"},{"id":55976718,"identity":"8b5b553f-56f4-4546-a4b5-05c1a3ed4ae2","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28883,"visible":true,"origin":"","legend":"\u003cp\u003eLactam-lactim tautomerism in alkaline medium.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/bf76069c9ba763db603a4128.png"},{"id":55976724,"identity":"ce25addf-8688-4845-aa8d-b63e56ebe711","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41379,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the experimental and theoretical curves for the Cu(II)/(UDP-GluA) system.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/479188aa4637113c0703c866.png"},{"id":55976717,"identity":"afe2db5e-9d50-47c8-8312-81bdb8a95ac8","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64771,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution diagram of the Cu(II)/(UDP-GluA) system (ratio1:1); C\u003csub\u003eCu\u003c/sub\u003e\u003csup\u003e\u003csub\u003e2+\u003c/sub\u003e\u003c/sup\u003e= C\u003csub\u003eL\u003c/sub\u003e= 1x10-3 mol/dm3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/ebe9f54543073863147a3d12.png"},{"id":55976720,"identity":"ed0aabd9-0527-4cbb-a7b2-14b73c7bb858","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79583,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis spectrum of the Cu(II)/(UDP-GluA) system.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/ebba1780a8d174fde8367060.png"},{"id":55976726,"identity":"21fedf2e-ba63-4fb6-af85-93945f433c65","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57313,"visible":true,"origin":"","legend":"\u003cp\u003eEPR spectra of: a) Cu(UDPGluA)H\u003csub\u003e2\u003c/sub\u003e, b) Cu(UDPGluA)H.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/653ddfb866c729e68168d9b8.png"},{"id":55976722,"identity":"1f5ebfc4-251d-4de4-83f2-5ccb991aad9a","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":254200,"visible":true,"origin":"","legend":"\u003cp\u003eCircular dichroism spectra of uridine-5’-diphosphoglucuronic acid in water at pH=3 (black line) and pH=10.0 (red line).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/9f30ac9436e2ae26e5e0a409.png"},{"id":55977221,"identity":"28e216ee-9c03-4093-81d6-b62dd33466c9","added_by":"auto","created_at":"2024-05-07 05:47:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":53394,"visible":true,"origin":"","legend":"\u003cp\u003eCircular dichroism spectra of the Cu(II) uridine-5’-diphosphoglucuronic acid system in water at pH=2.5 (black line), pH=5.0 (red line), pH=8.0 (green line) and pH=10.5 (blue line).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/3a40dcf55ea0d74ee31172ab.png"},{"id":55976719,"identity":"5e6864be-51c3-41cf-8dd7-a974ede4b388","added_by":"auto","created_at":"2024-05-07 05:39:07","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":29508,"visible":true,"origin":"","legend":"\u003cp\u003eLactam-lactim tautomerism in the uridine moiety and its derivatives [23]\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/0026d9cb7e1b7b16b21343da.png"},{"id":55977220,"identity":"522ef10e-d9dc-4d49-95b1-483d6242f0a3","added_by":"auto","created_at":"2024-05-07 05:47:07","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":24763,"visible":true,"origin":"","legend":"\u003cp\u003eCells viability after incubation with tested compounds for 24h and 72h in different pH.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/f239de8e6785a80011f7d118.png"},{"id":57201602,"identity":"5f1575b2-3a15-4d4a-a31e-e782bb6bc69c","added_by":"auto","created_at":"2024-05-27 10:03:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1212735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4184106/v1/678b328b-f73f-4ff7-979f-e60d18051ed6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thermodynamic studies of complexes in Cu(II)/Uridine- 5’-diphosphoglucuronic acid system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlycans are compounds that play an important role in living organisms. Changes in their concentration and differences in their structure on the surface of cells are important for diagnosing developing cancer or infection in the body. Compounds that regulate the level of glycans in the organism are sugar derivatives of uridine diphosphate. These derivatives also play a key role in the synthesis of glycans. Due to their properties, these compounds may constitute a new group of anticancer drugs [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetal ions belong to a group of elements that are essential for the proper functioning of organisms. They play an important role in the stabilization of protein structures and are an active center of many enzymes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. An important trace element in living organisms is the copper(II) ion, which can be found in the brain, liver, or bones. Copper(II) ions are part of many enzymes, including cytochrome c oxidase, peroxidase, tyrosinase, and lysil oxidase. Furthermore, these ions are involved in the synthesis of hemoglobin and melanin [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Copper(II) ion concentration disorders can cause diseases such as anemia, osteoporosis, Parkinson\u0026rsquo;s disease, hernias, Menkes syndrome, Hodgkin\u0026rsquo;s disease, leukemia, and general weakness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNucleic acids are biomolecules that play an important role in living organisms. These compounds are involved in the transcription, transmission, and storage of genetic information and also constitute the genetic material of a cell. Nucleotides are the basic unit that builds nucleic acids. A nucleotide consists of a nucleobase, five-carbon sugar (ribose or 2- deoxyribose), and a phosphate group. Depending on the origin of the amino acids, nucleotides can be divided into purine and pyrimidine nucleotides [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nucleosides, such as nucleotides, in their structure contain amino acids and sugar, ribose or deoxyribose. Both play a key role in the living organism: they participate in the synthesis of lipids and the metabolism of carbohydrates and are components of many coenzymes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One of the representatives of pyrimidine nucleosides is uridine, which is composed of sugar ribose and uracil. It is necessary in the process of RNA and glycogen synthesis and is a precursor of uridine phosphates. Uridine and its derivatives play an important role in the functioning of the central nervous system. Uridine-5'-triphosphate (UTP) is used during glycogen synthesis to produce uridine-5\u0026rsquo;-diphosphate glucose (UDP-glucose) [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNucleic acid bases can exist in several different forms of tautomerism. In molecules of nucleic acid bases, keto-enol and amino-imino tautomerism can occur. The formation of tautomeric forms is related to the presence of protons capable of migration in the molecules of these organic substances. The appearance of tautomeric forms is important, e.g. in pharmacy, chemistry, and physics. In addition, the formation of different tautomeric forms can influence the structural and chemical diversity of these compounds and thus their biological function. Therefore, replacing one nuclear base with its tautomer can result in an incorrect base pair, causing an error in the genetic code or a mutation [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eD-glucuronic acid (GluA) is a derivative of glucose and is classified as uronic acids. In its molecular components, it includes the carboxyl, hydroxyl, and pseudo-aldehyde group. D-glucuronic acid is obtained in the process of dehydrogenation of UDP-glucose [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These compounds play an important role in the glucuronidation process. This process allows the removal of compounds such as bilirubin, xenobiotics, and steroid hormones from the organism [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The presence of two functional groups, carboxyl and hydroxyl, in the D-glucuronic acid molecule promotes the formation of metal-ligand complex compounds. Ligands such as D-glucuronic acid can be used as masking agents for various metals, including toxic metals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUridine-5\u0026rsquo;-diphosphoglucuronic acid (UDP-GluA) is a derivative of uridine-5\u0026rsquo;-diphosphate and D-glucuronic acid and occurs in the liver and kidneys [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It\u0026rsquo;s synthesis is catalyzed by UDP-glucose dehydrogenase. Uridine-5\u0026rsquo;-diphosphoglucuronic acid plays an important role in reactions that are catalysed by UDP-glucuronosyltransferase. UDP-glucuronylotransferases are a group of enzymes involved in the detoxification process of the living organism. These enzymes catalyze the glucuronidation of potentially toxic and carcinogenic metabolic products such as cannabinoids and such compounds as thyroxin, some bile acids, morphine and acetaminophen. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe following article presents the results of potentiometric and spectroscopic studies on the formation of complex compounds in the system of uridine-5 '-diphosphoglucuronic acid and copper(II) ions. The internal coordination sphere in the obtained complexes was also determined. Furthermore, the antitumor properties of the obtained complexes were investigated.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBinary system of Copper(II) Ion/Uridine-5\u0026rsquo;-diphosphoglucuronic acid\u003c/h2\u003e \u003cp\u003eThe analysis of the potentiometric studies confirmed the presence of three protonated forms of the ligand: (UDP-GluA)H, (UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e, and (UDP-GluA)H\u003csub\u003e3\u003c/sub\u003e in the system studied. The protonation constants of these forms and their formation reactions are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The structural formula of the studied ligand with a highlighted potential coordination site is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn the pH range studied, the carboxylic group of D-glucuronic acid, the nitrogen atom in the uridine ring, and one of the phosphate residues are deprotonated and considered potential coordination centers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe protonation constant (log\u003cem\u003eβ\u003c/em\u003e) of uridine-5\u0026rsquo;-diphosphoglucuronic acid, the equilibrium constant of formation (log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e) (standard deviations are given in parentheses).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elog\u003cem\u003eβ\u003c/em\u003e; [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elog\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReaction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(UDP-GluA)H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.66(2); 9.40 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(UDP-GluA)\u003csup\u003e4\u0026minus;\u003c/sup\u003e + H\u003csup\u003e+\u003c/sup\u003e \u0026harr; (UDP-GluA)H\u003csup\u003e3\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.67(4); 12.79 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(UDP-GluA)H\u003csup\u003e3\u0026minus;\u003c/sup\u003e + H\u003csup\u003e+\u003c/sup\u003e \u0026harr; (UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(UDP-GluA)H\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.85(5); 14.19 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e + H\u003csup\u003e+\u003c/sup\u003e \u0026harr; (UDP-GluA)H\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe deprotonation of the -O-PO\u003csub\u003e3\u003c/sub\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e group (log\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.82, log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e=2.17) and the -COOH group (log\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.65, log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e=3.01) starts at a pH value lower than the study range. The partially protonated form occurs in the system up to a pH value of about 5,5. The deprotonation process of the proton of -N(3)H (log\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.64, log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e=8.64) begins below the test scale. This form is present in solution up to a pH of about 9.5 and at its maximum represents approximately 100% of all forms in the system. At pH above 9.5, the system is dominated by the fully deprotonated form of the ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt high pH values, when a proton from -N(3)H is deprotonated, lactam-lactim tautomerism occurs due to the alkaline medium. As a result of tautomerism, the free electron pair from the nitrogen atom migrate, causing the formation of a double bond between the C4 carbon atom and the nitrogen atom. The formation of the double bond causes an electron pair to leave the bond between the carbon atom C4 and the oxygen atom and migrates this pair to the oxygen atom (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComputer analysis of the potentiometric measurements confirmed the presence of two protonated forms and two hydroxocomplexes: Cu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e, Cu(UDP-GluA)H, Cu(UDP-GluA)(OH) and Cu(UDP-GluA)(OH)\u003csub\u003e3\u003c/sub\u003e. The stability constants (log\u003cem\u003eβ\u003c/em\u003e), equilibrium constants of formation (log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e), and examples of complex formation are presented in the table (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe overall stability constants (log\u003cem\u003eβ\u003c/em\u003e) and the equilibrium constants of formation (log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e) of the complexes formed in the studied system (standard deviations are given in parentheses).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elog\u003cem\u003eβ\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elog\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReaction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.61(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e + (UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e \u0026harr; Cu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.52(2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e + (UDP-GluA)H \u0026harr; Cu(UDP-GluA)H\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)(OH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.78(2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e + (UDP-GluA)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO \u0026harr; Cu(UDP-GluA)(OH)\u0026thinsp;+\u0026thinsp;H\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)(OH)\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-20.87(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCu(UDP-GluA)(OH)\u0026thinsp;+\u0026thinsp;2H\u003csub\u003e2\u003c/sub\u003eO \u0026harr; Cu(UDP-GluA)(OH)\u003csub\u003e3\u003c/sub\u003e + 2H\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe correctness of the assumed model was determined by comparing the theoretical computer-generated curve and the experimental curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The experimental and theoretical curves correspond practically over the entire range, which proves the correctness of the assumed model. The sigma parameter for the adopted model is: 12.405.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComplex Cu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e starts forming at a pH below 2.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This form dominates at pH value approximately 2.5 and binds about 55% of the cooper(II) ions. Cu(UDP-GluA)H started forming at pH value below 2.5. The protonated form dominates at pH 5.0-5.5 and binded maximally to 70% of copper(II) ions. The first hydroxy complex Cu(UDP-GluA)(OH) started to form at pH 5.5 and dominated at pH values about 8.0, where it binded approximately 65% of Cu\u003csup\u003e2+\u003c/sup\u003e. At pH 8.0, the las complex form started forming: Cu(UDP-GluA)(OH)\u003csub\u003e3\u003c/sub\u003e. This complex dominated at pH value above the test range. At pH about 10.5 this hydroxy complex binding about 55% of copper(II) ions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpectroscopic Studies\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eUV-vis and EPR spectroscopy\u003c/h2\u003e \u003cp\u003eSpectroscopic methods were used to analyze the forming complex compounds. UV-Vis and EPR measurements were performed at pH values that provided the highest possible percentage of a given form. These pH values were selected on the basis of distribution curves. The obtained spectroscopic parameters are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUV-Vis and EPR spectroscopic parameters for the formation of complex forms.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg\u003csub\u003eǁ\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003csub\u003eǁ\u003c/sub\u003e \u003c/p\u003e \u003cp\u003e[cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eλ\u003csub\u003emax\u003c/sub\u003e [nm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eε\u003c/p\u003e \u003cp\u003e[M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAbsorbance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003echromophore\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e136∙10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e{1O}\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145∙10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e{1O}\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)(OH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e{1N, 2O}\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA)(OH)\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e{1N, 3O}\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe change in the internal coordination sphere in complexes containing copper(II) ions is associated with a shift in absorbance toward lower wavelengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For the Cu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e and Cu(UDP-GluA)H complexes, coordination occurs through one oxygen atom derived from the glucuronic acid moiety or phosphate group. With increasing pH values, nitrogen in the UDP-GluA molecule is deprotonated and a coordination bond is formed with this donor atom. For the Cu(UDP-GluA)(OH) complex, coordination occurs through a nitrogen atom derived from the UDP molecule and two oxygen atoms derived from the glucuronic acid molecule and phosphate groups of the UDP molecule. The internal coordination sphere of the last complex form from the studied system includes a nitrogen atom N(3) derived from the UDP molecule and three oxygen atoms derived from carbonyl group -COOH of glucuronic acid and two phosphate residues -O-PO\u003csub\u003e2\u003c/sub\u003eH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the basis of EPR spectra analysis, the formation of monomeric complex forms in the studied system was confirmed, and characteristic spectra for copper(II) ions were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The spectral parameters g\u003csub\u003eǁ\u003c/sub\u003e = 2.39 and A\u003csub\u003eǁ\u003c/sub\u003e = 136∙10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the Cu(UDP-GluA)H\u003csub\u003e2\u003c/sub\u003e complex and g\u003csub\u003eǁ\u003c/sub\u003e = 2.37 and A\u003csub\u003eǁ\u003c/sub\u003e = 145∙10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the Cu(UDP-GluA)H complex indicate the participation of one oxygen atom in the formation of the coordination bond. The EPR spectra obtained are analogous due to the similar coordination mode in the compounds analysed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCD spectroscopy\u003c/h2\u003e \u003cp\u003eThe first series of CD measurements was carried on uridine-5\u0026rsquo;-diphosphoglucuronic acid at different pH to exclude protonation/deprotonation effect on the conformation of the ligand. Based on the results of potentiometric studies, pH 3.0 and 10.0 were chosen. The results of these measurements show that there is no significant change in CD spectra in the range of 250\u0026ndash;280 nm which originates from the absorption of uridine base. The maximum observed for the acidic solution is ∆ε\u0026thinsp;=\u0026thinsp;3.81 at 269 nm and ∆ε\u0026thinsp;=\u0026thinsp;4.29 at 266 for the basic one. In the short-wave part of the spectra a negative Cotton effect can be observed for the sample in the basic solution. This result is usually related [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] to deprotonation of chiral carboxylic acid, which shifts the n-π* transition and CD maxima towards shorter wavelengths (∆ε = -0.84 at 228 for the acidic solution nm and ∆ε = -2.14 at 216 for the basic one). The loaded CD spectra for both solutions are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and indicate the conformation of uridine-5\u0026rsquo;-diphosphoglucuronic acid is not pH dependent in water solutions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the uridine-5\u0026rsquo;-diphosphoglucuronic acid have many potential electron donor sites the possible coordination of Cu(II) ions may take place by different parts of the ligand causing its conformational changes. These coordination modes may also vary due to pH changes. The CD measurements of Cu(II)/UDP-GluA system were performed at a pH that was previously determined on the basis of potentiometric measurements knowing that the main form of the complex is usually present alongside other minor ones. In the case of measurements for all pH, the same sequence of Cotton effects is observed with a positive long-wave effect located in the range of 262\u0026ndash;272 nm and negative Cotton effects located in the range of 218\u0026ndash;239 nm. The main Cotton effects at a given wavelength are summarized in the Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCotton effects for Cu(II)/UDPGluA system in water solutions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e∆ε (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.98 (272)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.91 (267)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.85 (267)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.21 (262)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.95 (239)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.85 (239)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.70 (239)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.98 (237)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-1.01 (224)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-1.11 (228)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.19 (227)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.94 (227)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-1.15 (219)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.50 (219)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-2.70 (218)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results indicates no substantial changes in the ligand conformation in the pH range of 2.5-8.0. The spectrum measured at pH\u0026thinsp;=\u0026thinsp;10.5 has more intense and blue shifted maximum which is in agreement with NMR shifts measurements and may be associated with the change of donor atoms in the system. Additionally this change can be also connected with the shift of equilibrium of base-promoted lactam-lactim tautomerization, which is also observed in the shift of C4 signal in the \u003csup\u003e13\u003c/sup\u003eC NMR spectra. The negative Cotton effects in the range of 218\u0026ndash;239 nm are derived from the interaction of the carboxylic acid residue with the chiral environment. As expected, there is a general tendency to increase the intensity of these effects and shift towards shorter wavelengths with increasing pH, however, the mutual proximity of positive Cotton effects of relatively high intensity may affect their height and location on the CD spectrum, which makes their precise interpretation difficult. CD spectra of the Cu(II)/UDP-GluA system are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNMR spectroscopy\u003c/h2\u003e \u003cp\u003eNMR studies were conducted to determine the mode of coordination in the studied system. The studies were performed for ligand and complexes for two pH values. The results obtained are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eDifferences between \u003csup\u003e13\u003c/sup\u003eC NMR and \u003csup\u003e31\u003c/sup\u003eP NMR chemical shifts form the ligand in the Cu(UDP-GluA) system in relation to the free ligand [ppm].\u003c/p\u003e\n\u003ctable style=\"width: 5.0e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.4pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003eSystem\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.15pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003epH\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"17\" style=\"width: 431.5pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;vertical-align:baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003e(UDP-GluA)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC1\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC2\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC3\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC4\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC5\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC6\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC7\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC8\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC9\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC10\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eC11\u0026rsquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eP1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(127, 127, 127);padding: 0in 5.4pt;height: 19.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003eP2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.4pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003eCu(UDP-GluA)H\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.15pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e5.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e0.06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e-0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e0.06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.47\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:red;\"\u003e0.20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border: none;padding: 0in 5.4pt;height: 48.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:red;\"\u003e7.93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.4pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;\"\u003eCu(UDP-GluA)(OH)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.15pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;vertical-align:baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e8.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e-0.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:red;\"\u003e-0.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e-0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;\"\u003e0.07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:red;\"\u003e-0.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.38\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.8pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.95pt;border-top: 1pt solid rgb(127, 127, 127);border-left: none;border-bottom: 1pt solid rgb(127, 127, 127);border-right: none;padding: 0in 5.4pt;height: 56.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:5.65pt;margin-bottom: 0in;margin-left:5.65pt;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:normal;vertical-align: baseline;'\u003e\u003cspan style=\"font-size:13px;color:red;\"\u003e4.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n \u003cp\u003eAnalysis of \u003csup\u003e13\u003c/sup\u003eC NMR and \u003csup\u003e31\u003c/sup\u003eP NMR spectra revealed changes in chemical shifts between the free ligand and the complex compound. At low pH values, changes in chemical shifts were observed in carbon C6\u003csup\u003e\u0026rsquo;\u003c/sup\u003e (0.20) derived from the glucuronic acid moiety and at the phosphorus atom P2 (7.93). At these pH values, coordination occurs between the oxygen atom of glucuronic acid or the oxygen atom of the phosphate group of the UDP moiety. This type of coordination occurs for the complex Cu(UDP-GluA)H. At higher pH values, changes in chemical shifts were observed at the C4 (-0.91) carbon atom and P2 (4.5) phosphorus atom of the UDP molecule and C6\u003csup\u003e\u0026rsquo;\u003c/sup\u003e (-0.70) carbon atom of glucuronic acid moiety. These changes are due to deprotonation of nitrogen atom at higher pH values and the occurrence of lactam-lactim tautomerism. These changes explain the chemical shift of only the C4 carbon and not the C4 and C2 carbons in the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum. At pH 8.0, coordination occurs through an oxygen atom located at the C4 carbon in the uridine ring and through one of the oxygen atoms of the phosphate group and an oxygen atom derived from the carbonyl group of D-glucuronic acid. A schematic of the lactam-lactim tautomerism is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity\u003c/h2\u003e \u003cp\u003eThe IC50 (half maximal inhibitory concentration) is defined as the concentration of compound needed to inhibit a biological process or response by 50%. The metabolic activity of the cells after 24 h incubation with tested compounds at pH5 was similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The same for IC50 values (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However after 72h higher cytotoxicity could be observed for Cu UDP-GluA. A549 cells reduced their activity by 44,4% in comparison to control, e.g. cells cultured in standard growth medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn turn, after 72h incubation with UDP-GluA at pH8 no changes in cells\u0026rsquo; metabolic activity could be detected, therefore IC50 value could not be calculated. Cu(UDP-GluA) at pH8 after 72h incubation reduced the A459 cell\u0026rsquo;s metabolic activity by 52,2%. However, the effect was strongly time-dependent.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC50 values [uM] calculated based on the MTT assay results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(UDP-GluA) pH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.317\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.781\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA) pH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.377\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.116\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(UDP-GluA) pH8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.295\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu(UDP-GluA) pH8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.719\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.094\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003en.d. \u0026ndash; not detectable, to low cytotoxicity to calculate LC50.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe formation of complexes in the binary system of uridine-5'-diphosphoglucuronic acid and copper(II) ions has been established. The existence of two types of complex forms in the system was observed: MHxL and ML(OH)x. Depending on the pH, different coordination modes were observed in the obtained complex compounds. The types of chromophores were determined by UV-Vis, EPR and NMR spectroscopic studies. It was observed that, at low pH values, coordination occurs through an oxygen atom derived from the glucuronic acid molecule or phosphate group. As the pH increases, the nitrogen atom in the ligand molecule is deprotonated and a coordination bond is formed with this donor atom. Oxygen atoms derived from UDP phosphate residues also play an important role in the coordination process. The bioassays carried out showed an increase in the biological activity of the complex compounds tested against the free ligand. As the incubation time increases, the biological activity of the tested compounds increases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eUridine-5\u0026rsquo;-diphosphoglucuronic acid trisodium salt was obtained from Sigma-Aldrich and copper (II) nitrate was obtained from Merck. All of these materials were used without purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePotentiometric study\u003c/h2\u003e \u003cp\u003ePotentiometric titration was performed using a Titrando 713 Methrom equipped with an autoburette with a Methrom 6.0233.100 combined glass electrode calibrated prior to each test. Calibration was performed prior to each titration with two buffer solutions of pH 4.002 and pH 9.225. All measurements were carried out under strictly defined conditions of constant ionic strength of 0.1 M KNO\u003csub\u003e3\u003c/sub\u003e, temperature 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, an inert gas atmosphere helium (He 5.0 Ultra High Purity; 9.1 m\u003csup\u003e3\u003c/sup\u003e; 200 bar) and a pH range of 2.5 to 11.0. The metal to ligand ratio was 1:1 and the concentration of ligands and copper(II) ions [Cu\u003csup\u003e2+\u003c/sup\u003e] was 0.001 mol/dm\u003csup\u003e3\u003c/sup\u003e. The protonation constant and the stability constant of the complex were determined using the HYPERQUAD 2008 programme. The calculations allowed us to determine the model of complex formation in the systems studied. The correctness of the assumed model was verified by analysing the standard deviations and the convergence of the experimental and theoretical curves [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. At a pH of around 8, each sample began to take on a light blue colour, and a small amount of precipitate was observed when the samples were discarded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eUV-vis spectroscopy\u003c/h2\u003e \u003cp\u003eUV-Vis spectroscopy studies were performed on the SHIMADZU UV-1900 spectrophotometer using the UVProbe programme. Measurements were made in the wavelength range of 550 to 900 nm. The concentration of the metal ion was 0.001 mol/dm\u003csup\u003e3\u003c/sup\u003e and the metal to ligand molar ratios were 1:1. The data obtained with UVProbe were saved as a text file, and the UV-Vis spectra were then produced with SigmaPlot 11.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEPR spectroscopy\u003c/h2\u003e \u003cp\u003eThe EPR spectra were carried out at a temperature of -196\u0026deg;C, using glass capillary tubes, and recorded on an SE/X2457 Radiopan spectrometer. EPR studies were performed for copper (II) ion systems, in which the concentration of metal ions was 0.005 M in a solution of water:glycol 3:1 and the metal ligand ratio was 1:1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNMR spectroscopy\u003c/h2\u003e \u003cp\u003e \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e31\u003c/sup\u003eP NMR spectroscopic investigations were performed for the ligand and complex forms formed at two pH values: pH\u0026thinsp;=\u0026thinsp;5.0 and pH\u0026thinsp;=\u0026thinsp;8.0. Samples were prepared by dissolving the corresponding reactants in a deuterated solvent and the pD values of each prepared sample were determined by NaOD and DCl taking into account the relation pD\u0026thinsp;=\u0026thinsp;pH\u0026thinsp;+\u0026thinsp;0.4 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The concentration of ligands in the samples was 0.05 mol/dm\u003csup\u003e3\u003c/sup\u003e and the M:L ratio was 1:100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCD spectroscopy\u003c/h2\u003e \u003cp\u003eThe CD and corresponding UV spectra were recorded on a JASCO J810 spectropolarimeter at ambient temperature. Spectra were recorded in the range of 185\u0026ndash;400 nm in water solutions and accumulated with four scans for both UDPGluA and its copper(II) complexes. Water for the experiments was extra purified by Merck Millipore Simplicity UV apparatus to lower the absorbance especially in the short-wave part of the measuring range. The measurements were performed in N\u003csub\u003e2\u003c/sub\u003e gas atmosphere (flow 10 L/min) and optical pathlength was 0.1 cm. Concentrations of measured solutions were 1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M, which allowed the absorbance to be maintained at an acceptable level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell line and cell culture\u003c/h2\u003e \u003cp\u003eThe A549 cell line was initially initiated through an explant culture of lung carcinoma tissue from a 58-year-old male. The cell line was purchased from ATCC: the Global Bioresource Center. Cells are hypotriploid with the modal chromosome number of 66 in 24% of cells. As verified at ATCC, there are six markers present in single copies in all cells, e.g. der(6)t(1;6) (q11;q27); del(6) (p23); del(11) (q21), del(2) (q11), M4 and M5. Most cells have doubled sex chromosomes, single copies of N2 and N6 chromosomes, and four copies of N12 and N17 chromosomes.\u003c/p\u003e \u003cp\u003eBasic cell culture reagents and other materials were purchased from Corning. A549 cells were routinely cultured in F-12K medium supplemented with a fetal bovine serum to a final concentration of 10%. They were maintained at 37\u0026deg;C in a humidified cell culture incubator (Thermo Fisher Scientific) with 5% CO\u003csub\u003e2\u003c/sub\u003e at a concentration between 6x10\u003csup\u003e3\u003c/sup\u003e and 6x10\u003csup\u003e4\u003c/sup\u003e cells per cm\u003csup\u003e2\u003c/sup\u003e. The standard growth medium was changed two or three times a week.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eThe assay allows for the detection of the cells\u0026rsquo; metabolic activity. It is based on the intracellular reduction of the water-soluble MTT reagent to an insoluble formazan by respiring cells. A549 cells were detached from the growth surface, counted and transferred to 96-well plates at a density of 4.5x10\u003csup\u003e3\u003c/sup\u003e cells per cm\u003csup\u003e2\u003c/sup\u003e. After 24h preincubation in standard conditions, tested compounds were added, and cells were cultured for 24 or 72 h. The medium was discarded, and cells were incubated with the MTT solution (1mg/ml) for 1 h at 37\u0026deg;C. Formed purple formazan crystals were dissolved in DMSO (StanLab). The absorbance was read at OD 570 nm using a microplate reader Multiskan Sky (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":" \u003ch2\u003eAuthor contributions statement\u003c/h2\u003e \u003cp\u003eConceptualization, K.S. and M.Z.; methodology, K.S., M.Z., J.G. and R.J; validation, M.Z and R.J.; formal analysis, M.Z. and J.G.; investigation, K.S., J.G., A.T. and A.B.; writing\u0026mdash;original draft preparation, K.S.; visualization, K.S.; supervision, R.J., M.Z. and K.S. All authors reviewed the manuscript.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, K.S. and M.Z.; methodology, K.S., M.Z., J.G. and R.J; validation, M.Z and R.J.; formal analysis, M.Z. and J.G.; investigation, K.S., J.G., A.T. and A.B.; writing\u0026mdash;original draft preparation, K.S.; visualization, K.S.; supervision, R.J., M.Z. and K.S. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA., Varki, Glycobiology, 2017, 27, 1, 3-49, doi: 10.1093/glycob/cww086\u003c/li\u003e\n\u003cli\u003eFuster, M. M., \u0026amp; Esko, J. D. (2005). The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 5(7), 526-542. doi:10.1038/nrc1649\u003c/li\u003e\n\u003cli\u003eDube, D. H. \u0026amp; Bertozzi, C. R. (2005). Glycans in cancer and inflammation - Potential for therapeutics and diagnostics. Nature Reviews Drug Discovery, 4(6), 477\u0026ndash;488. doi:10.1038/nrd1751\u003c/li\u003e\n\u003cli\u003eSchachter, H., Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides, Biochemistry and Cell Biology, 64(3), 163\u0026ndash;181, 1986\u003c/li\u003e\n\u003cli\u003eDennis J., W., Nabi I., R., Demetriou M., Metabolism, Cell Surface Organization, and Disease, Cell, 139(7): 1229\u0026ndash;1241, 2009\u003c/li\u003e\n\u003cli\u003eBrewer C., F., Miceli M., C., Baum L., G., Clusters, bundles, arrays, and lattices: novel mechanisms for lectin\u0026ndash;saccharide- mediated cellular interactions, Current Opinion in Structural Biology, 12(5), 616\u0026ndash;623, 2002\u003c/li\u003e\n\u003cli\u003eMoustakas, M. The role of metal ions in biology, biochemistry and medicine, Materials, 2021, 14, 549, 2-4, doi: 10.3390/ma14030549\u003c/li\u003e\n\u003cli\u003eOves, M., Saghir Khan, M., Huda Quari, A., Nadeen Felemban, M., Almeelbi, T., Journal of Bioremediation and Biodegradation, 2016, 7, 2, 1-15, doi: 10.4172/2155-6199.1000334\u003c/li\u003e\n\u003cli\u003eAngelova, M., Asenova, S., Nedkova, V., Koleva-Kolarova, R., Tarika Journal of Science, 2011, 9, 88-98\u003c/li\u003e\n\u003cli\u003eOsredkar, J., Sustar, N., Journal of Clinical Toxicology, 2011, 1-18, doi:10.4172/2161-0494.S3-001 \u003c/li\u003e\n\u003cli\u003eHoffmann, S., K., Goslar, J., Bregier-Jarzebowska, R., Gasowska, A., Zalewska, A., Lomozik,L., Journal of Inorganic Biochemistry, 2017, 177, 89-100, doi: 10.1016/j.jinorgbio.2017.09.004 \u003c/li\u003e\n\u003cli\u003eHorodyjewska, A., Popioek,., Kocot, J., Biometals, 2014, 27, 611-621, doi: 10.1007/s10534-014-9736-5\u003c/li\u003e\n\u003cli\u003eKaczkowski, J., Podstawy Biochemii, Wydawnictwo naukowe PWN, Warszawa, 2017 \u003c/li\u003e\n\u003cli\u003eKumar, A., Kumar, D., Journal of Molecular Structure, 2020, 1222, 1-12, doi.org/10.1016/j.molstruc.2020.128889\u003c/li\u003e\n\u003cli\u003eBelmont, P., Constant, J., F., Demeunynck, M., The Royal Society of Chemistry, 2001, 30, 70-81, doi.org/10.1039/A904630E\u003c/li\u003e\n\u003cli\u003eLomozik, L., Jastrzab, R., Journal of Solution Chemistry, 2007, 36, 357-374, doi: 10.1007/s10953-006-9114-1\u003c/li\u003e\n\u003cli\u003eSaenger, W., Angewandte Chemie International Edition, 1973, 12, 591-682, doi.org/10.1002/anie.197305911 \u003c/li\u003e\n\u003cli\u003eYamamoto, T., Koyama, H., Kurajoh, M., Shoji, T., Tsutsumi, Z., Moriwaki, Y., Clinica Chimica Acta, 2011, 412, 1712-1724, doi.org/10.1016/j.cca.2011.06.006\u003c/li\u003e\n\u003cli\u003eDobolyi, A., Juhasz, G., Kovacs, Z., Kardos, J., Current Topics in Medicinal Chemistry, 2011, 11, 1058-1067, doi: 10.2174/156802611795347618\u003c/li\u003e\n\u003cli\u003eCansev, M., Brain Research Reviews,2006, 52, 389-397, doi.org/10.1016/j.brainresrev.2006.05.001\u003c/li\u003e\n\u003cli\u003eBerg, J., M., Tymoczko, J., L., Stryer, L., Biochemistry, W.H. Freeman and Company, 2002 \u003c/li\u003e\n\u003cli\u003eZhang, N., Yin, Y., Xu, JS, Chen, WS, Molecules, 2008, 13, 1551-1569, doi: 10.3390/molecules13081551\u003c/li\u003e\n\u003cli\u003eWojnarowska, Z., Paluch, M., Wlodarczyk, P., Dulski, M., Wrzalik, R., Roland, C., M., The Journal of Pchysical Chemistry Letters, 2012, 3, 2288-2292, doi: 10.1021/jz300541t\u003c/li\u003e\n\u003cli\u003eFedeles, B., I., Li, D., Singh, V., Frontoers in Molecular Biosciences, 2022, 8, 1-13, doi: 10.3389/fmolb.2021.823253\u003c/li\u003e\n\u003cli\u003eSingh, V., Fedeles, B., I., Essigmann, J., M., Role of Tautomerism inRNA Biochemistry, 2014, 21(1), 1-13, doi: 10.1261/rna.048371.114\u003c/li\u003e\n\u003cli\u003eVina, I., Linde, R., Patetko, A., Sejmonows, P., IJRRAS, 2013, 14, 2, 217-230, www.arpapress.com/Volumes/Vol14Issue2/IJRRAS_14_2_02.pdf \u003c/li\u003e\n\u003cli\u003eDutton, G., J., Glucuronic Acid Free and Combined, Academic Press New York and London, 1966 \u003c/li\u003e\n\u003cli\u003eJastrzab, R., Nowak, M., Skrobanska, M., Zabiszak, M., Journal of Coordination Chemistry, 2016, 1-19, doi: 10.1080/00958972.2016.1205738\u003c/li\u003e\n\u003cli\u003eVina, I., Sejmonows, P., Linde, R., Patetko, A., IJRRAS, 2013, 14, 1, 17-25, www.arpapress.com/Volumes/Vol14Issue1/IJRRAS_14_1_02.pdf \u003c/li\u003e\n\u003cli\u003eRichel, A., Laurent, P., Wathelet, B., Wathelet, J., Paquot, M., Tetrahedrol Letters, 2010, 51, 1356-1360, doi.org/10.1016/j.tetlet.2009.12.065\u003c/li\u003e\n\u003cli\u003eFerrari, E., Grandi, R., Lazzari, S., Saladini, M., Journal of Inorganic Biochemistry, 2005, 99, 2381-2386, doi: 10.1371/journal.pone.0057937\u003c/li\u003e\n\u003cli\u003eCapiello, M., Giuliani, L., Rane, A., Pacifici, G., M., European Journal of Drug and Pharmacokinetics, 2000, 25, 161-164, doi: 10.1007/BF03192308\u003c/li\u003e\n\u003cli\u003eHauser, S., C., Ziurys, J., C., Gollan, J., L., Biochemica et Biophysica Acta, 1988, 967, 149-157, doi: 10.1016/0304-4165(88)90004-9\u003c/li\u003e\n\u003cli\u003eJastrzab, R., Lomozik, L., Coordination mode in the binary systems of copper(II)/O-phospho-L-serine. Journal of Coordination Chemistry, 2009, 62(5), 710\u0026ndash;720. doi:10.1080/00958970802317855 \u003c/li\u003e\n\u003cli\u003eGlasoe, P.K.; Long, F.A. Use of glass electrodes to measure acidities in deuterium oxide. J. Phys. Chem. 1960, 64, 188\u0026ndash;190, doi:10.1021/j100830a521.\u003c/li\u003e\n\u003cli\u003eE., Garribba, E., Lodyga-Chruscinska, D., Sanna, G., Micera, Inorganica Chimica Acta, 2001, 322(1-2), 87-98, doi:10.1016/s0020-1693(01)00554-0\u003c/li\u003e\n\u003cli\u003eKacprzak, K., Grajewski, J., Gawronski, J., Tetrahedron: Asymetry; Indicator displacement sensor for efficient determination of \u0026alpha;-hydroxydicarboxilic acids and their chiral discrimination; 2006, 17 (9), 1332-1336; doi: 10.1016/j.tetasy.2006.03.037\u003c/li\u003e\n\u003cli\u003eHoffmann, M., Grajewski, J. Gawronski, J., New Journal of Chemistry, Extending the applications of circular dichroism in structure elucidation: aqueous environment breaks the symmetry of tartrate dianion, 2010, 34 (9), 2020-2026, doi: 10.1039/C0NJ00072H\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"copper(II) ions, uridine derivatives, potentiometric measurements, spectroscopic studies, biological studies","lastPublishedDoi":"10.21203/rs.3.rs-4184106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4184106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBinary system of uridine-5'-diphosphoglucuronic acid with copper(II) ions have been studied. Potentiometric studies in aqueous solutions using computer data analysis were carried out. The pH of dominance, the overall stability constants (log\u003cem\u003eβ\u003c/em\u003e) and the equilibrium constants of the formation reaction (log\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e) were determined for each complex compound formed in the studied system. Spectroscopic studies were carried out to determine the mode of coordination in the compounds studied. Biological analysis of the compounds obtained showed an increase in the biological activity of the complexes tested against the free ligand.\u003c/p\u003e","manuscriptTitle":"Thermodynamic studies of complexes in Cu(II)/Uridine- 5’-diphosphoglucuronic acid system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 05:39:02","doi":"10.21203/rs.3.rs-4184106/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31300816-93f4-42f6-965a-8a16e1b91ffa","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31436667,"name":"Biological sciences/Biochemistry/Bioinorganic chemistry"},{"id":31436668,"name":"Biological sciences/Biochemistry/Biophysical chemistry"},{"id":31436669,"name":"Biological sciences/Biochemistry/Chemical modification"},{"id":31436670,"name":"Biological sciences/Biochemistry/Dna"},{"id":31436672,"name":"Biological sciences/Biochemistry/Metals"},{"id":31436674,"name":"Biological sciences/Biochemistry"},{"id":31436676,"name":"Biological sciences/Chemical biology"},{"id":31436678,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2024-05-27T09:55:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 05:39:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4184106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4184106","identity":"rs-4184106","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}